Antineoplastic Agents. 340. Isolation and Structural Elucidation of

Separation of two trace cancer cell growth inhibitory (P388 leukemia) fractions from about. 1000 kg of wet Gulf of Mexico Bugula neritina (Bryozoa) ha...
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J. Nat. Prod. 1996, 59, 286-289

Antineoplastic Agents. 340. Isolation and Structural Elucidation of Bryostatins 16-181 George R. Pettit,* Feng Gao, Peter M. Blumberg,† Cherry L. Herald, John C. Coll,‡ Yoshiaki Kamano, Nancy E. Lewin, Jean M. Schmidt, and Jean-Charles Chapuis Cancer Research Institute and Department of Chemistry, Arizona State University, Tempe, Arizona 85287-1604 Received September 20, 1995X

Separation of two trace cancer cell growth inhibitory (P388 leukemia) fractions from about 1000 kg of wet Gulf of Mexico Bugula neritina (Bryozoa) has led to the isolation of bryostatins 16-18 (2-4). A combination of HRFABMS and high-field (400 MHz) 1H- and 13C-NMR spectral analyses were employed to assign the structures. The three new 20-desoxybryostatins 16 (2), 17 (3), and 18 (4) showed significant growth inhibitory activity (P388 ED50, 2, 9.3 × 10-3 µg/ mL, 3, 1.9 × 10-2 µg/mL, and 4, 3.3 × 10-3 µg/mL) against murine P388 lymphocytic leukemia. The marine bryozoan Bugula neritina L. (family Bugulidae) has proved to be an exciting and exceptionally useful source of new anticancer drugs of the bryostatin class.2 From our initial observations3 in 1968 that B. neritina offered promise of containing very potent antineoplastic constituents, we proceeded (1968-1981) to isolate and determine the structures of bryostatins 1-15 and bryostatins A and B.2,4-10 After the isolation and structural determination of bryostatin 1 (1) in 1981, preclinical development was undertaken and the first phase I human clinical trials began in early 1991. Promising initial clinical results have been reported.11-14 Currently, the U.S. National Cancer Institute has entered bryostatin 1 into phase II clinical trials. Because of the encouraging clinical progress of bryostatin 1 and the considerable utility in employing the bryostatins as biochemical probes,15-22 we have continued to study structural modifications23,24 and isolate new members10 of this important series.2,4-10,25 Since Gulf of Mexico (Florida) collections of B. neritina4 have been found to afford the greatest variety of new bryostatins, we further explored the trace components of a 1986 recollection (1000 kg, wet wt) employing assays against the murine P388 lymphocytic leukemia cell line to guide the separation. From a complex fraction containing bryostatins 4-8,4,5,6 bryostatin 10,8 and bryostatin 14,10 we have succeeded in isolating the new trace cancer cell growth inhibitory constituents designated bryostatins 16 (2), 17 (3), and 18 (4) (Chart 1). Successive column chromatographic separations using Sephadex LH-20 and silica gel followed by high speed countercurrent distribution26 led to two penultimate fractions. The first of these was separated by HPLC on silica gel and found to contain bryostatin 16 (2) accompanied by bryostatins 4-8 and bryostatin 17. Separation of the second fraction was achieved by normal-phase silica gel semipreparative HPLC to give bryostatin 18 accompanied by bryostatins 4-8 and 10. Bryostatins 16 (2) and 17 (3) proved to be stereoisomers. Mass spectra of the two isomers led to the same molecular formula. Structural elucidation of the two † Molecular Mechanisms of Tumor Promotion Section, National Cancer Institute, 37 Convent Dr., MSC 4255, Bethesda, MD 20892-4255. ‡ University College of Central Queensland, Rickhampton M.C., Queensland 4702, Australia. X Abstract published in Advance ACS Abstracts, February 1, 1996.

0163-3864/96/3259-0286$12.00/0

isomers was based primarily on careful analysis of the high-field (400 MHz) 2D NMR spectra. From the 1HNMR data, significant differences were exhibited by the signals corresponding to H-20, H-22a, and H-22b (see Table 1). The dramatic downfield shift of the H-20 proton signal from δ 5.50 in bryostatin 16 to δ 6.78 in bryostatin 17 was attributed to proximity of a deshielding carbonyl oxygen. The H-22a proton of bryostatin 16 was found at δ 3.57 compared to the H-22a chemical shift at δ 2.48 in bryostatin 17. Otherwise, the remaining 1H-NMR signals of bryostatins 16 and 17 were very similar. When the 13C-NMR spectra of the two compounds were compared, the chemical shift for the C-20 of bryostatin 16 was δ 101.55 vs. 97.14 for bryostatin 17 and for C-22 was δ 32.82 vs. 37.10, respectively (Table 2). On the basis of 1H-NMR spectral data, the bryostatin 16 (2) C-21, 34 double bond was assigned the same geometry as in bryostatin 1 and the opposite geometry for bryostatin 17 (3). Apparently, the C-35 carbonyl group of bryostatin 17 resides closer to the C-20 proton than in the case of bryostatin 16. An overall interpretation of the 1H - and 13C-NMR data (Tables 1 and 2) combined with the NOE and HRFAB mass spectral data showed that bryostatin 16 was the ∆19,20derivative of bryostatin 10 (5)8 and bryostatin 17 was the ∆19,20-derivative of bryostatin 18 (see below). While it is conceivable that both bryostatin 16 and 17 were formed from bryostatins 10 and 18, respectively, during the isolation procedures, this is unlikely in view of the usual good stability of the bryostatin C-19 hydroxyl group. The HRFABMS data from bryostatin 18 (4) indicated the molecular formula C42H64O15. The low resolution FABMS indicated ready loss of water by the appearance of ions at m/z 815 [M + Li]+(100) and 797 [M + Li H2O]+(42). The molecular formula of bryostatin 18 corresponded to that of bryostatin 10 (5). Thus, the detailed structure of bryostatin 18 was determined by direct comparison of its NMR spectra with those of bryostatin 10.8 While both the 1H- and 13C-NMR spectra were very similar, some differences were obvious. In the 1H-NMR spectrum of bryostatin 18, the two H-20 proton signals were at δ 3.79 and 2.10 and the two from H-22 were at δ 2.23 and 2.02. By comparison, the H-20 protons of bryostatin 10 appear at δ 2.37 and 2.20 and signals for the two H-22 protons at δ 4.00 and 1.95. In addition, the 13C-chemical shifts for the C-20

© 1996 American Chemical Society and American Society of Pharmacognosy

Notes

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Chart 1

and C-22 carbons of bryostatin 18 (4) appeared at δ 33.70 and 43.13 while the C-20 and C-22 carbon atoms of bryostatin 10 were assigned, respectively, δ 40.39 and 36.63 (Tables 1 and 2). Therefore, bryostatin 18 was assigned the structure 4 corresponding to the C-21 f C-34 geometrical isomer of bryostatin 10. The discovery of bryostatins 16-18 proved to be useful for a variety of reasons. Foremost among these was the opportunity to further assess limits of the structural pharmacophore proposed for the bryostatin effects on protein kinase C.27 Loss of the C-19 hydroxyl group led to a 100-fold decrease in PKCR activity by bryostatins 16 and 17 compared with bryostatin 10, while the geometry of the isomers (cf. bryostatins 16 and 17) made little difference. Furthermore, it permitted experimental evaluation of the role of the C-9 hydroxyl group in the phorbol ester pharmacophore.27-29 In contrast to the availability of derivatives at the other oxygens, elimination of the C-9 oxygen has so far been

synthetically inaccessible in phorbol and interpretation has been further clouded by potential hydrogen bonding between the C-9 hydroxyl and the carbonyl of the phorbol 13-esters. Modeling indicates that the C-9 hydroxyl of phorbol is homologous to the C-19 hydroxyl of the bryostatins.28 Binding of bryostatins 16 and 17 to protein kinase C R, assayed under our usual conditions in the presence of phosphatidylserine/Triton X-100,30 yielded Ki values of 118 ( 2 nM (n ) 3) and 188 ( 7 nM (n ) 3), respectively. The latter value is in good agreement with our previous bryostatin findings.31 For comparison, bryostatin 10 yielded a Ki of 1.56 ( 0.16 nM (n ) 3) and bryostatin 18 yielded a Ki of 4.82 ( 0.06 nM (n ) 3); the value for bryostatin 10 is in good agreement with previous measurements.27-32 We conclude that (a) the configuration of the C-21 f C-34 double bond has a rather modest influence on binding affinity, with slightly higher affinity for the bryostatin 1 configuration, and (b) the C-19 hydroxyl contributes

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Table 1. 1H-NMR (400 MHz) Assignments (δ, ppm) for Bryostatins 16-18 in Deuterioacetonitrile Solution with Tetramethylsilane as Reference position

bryostatin 16

bryostatin 17

2a 2b 3 4a 4b 5 6a 6b 7 10a 10b 11 12a 12b 14a 14b 15 16 17 20a 20b 22a 22b 23 24a 24b 25 26 27 28 29 30 32 33 34 36 37 OR 3′-5′ 19OH

2.47 * 2.47 * 4.16 m 1.64 m 1.64 m 4.12 m 1.67 m 1.40 dt (12, 12) 5.10 dd (5, 12) 1.86 * 1.78 dd (3, 15) 3.78 * 2.23 * 2.18 * 3.68 * 1.89 * 3.98 brdd (7.3, 12) 5.41 dd (7.3, 16) 5.92 d (16) 5.50 brs

2.61 brd (16) 2.38 dd (6, 16) 4.18 m 1.52 brdd (11,12) 1.47 brdd (12,12) 3.70 brt (12) 1.64 brdd (5, 12) 1.40 dt (12, 12) 4.89 dd (5, 12) 2.25 dd (2, 15) 1.75 dd (11, 15) 3.95 brt (11) 2.26 brd (15) 2.17 brdd (11, 15) 3.70 brd (12) 2.02 brt (12) 4.00 brdd (8, 12) 5.54 dd (8, 16) 6.12 d (16) 6.78 brs

Notes Table 2. 13C NMR Assignments (δ, ppm at 100 MHz) for Bryostatins 10, and 16-18 in Deuterioacetonitrile Solution with Tetramethylsilane as Internal Standard

bryostatin 18

3.57 brd (17) 2.25 brdd (11, 17) 3.82 brt (11) 1.98 brt (11) 1.88 m 5.20 brd (11) 3.76 * 1.10 d (6.6) 0.97 s 0.89 s 5.75 brs 1.21 s 1.19 s 5.47 brs 3.66 s 3.61 s

2.50 brs 2.50 brs 4.04 m 1.80 * 1.70 * 4.20 brt (12) 1.64 * 1.45 dt (12, 12) 5.07 dd (5, 12) 1.99 * 1.73 * 3.92 * 2.17 * 2.17 * 3.57 brd (12) 1.91 * 4.10 * 5.31 dd (8, 16) 5.87 d (16) 3.79 d (12) 2.10 * 2.48 brdd (11, 15) 2.23 * 2.32 brd (15) 2.02 * 3.87 brt (11) 3.96 * 1.96 * 1.95 * 1.85 m 1.62 m 5.35 brd (11) 5.12 * 3.75 dq (6.7, 6.7) 3.75 * 1.10 d (6.7) 1.07 d (6.7) 0.98 s 1.02 s 0.87 s 1.01 s 5.72 brs 5.76 brs 1.21 s 1.09 s 1.29 s 0.89 s 5.25 brs 5.73 brs 3.65 s 3.66 s 3.62 s 3.63 s

1.16 s

1.16 s

1.16 s 4.74 d (3)

*Couplings for these signals were obscured.

a factor of 100 to the binding affinity, consistent with its forming an element of the pharmacophore. A further conclusion is that not all elements of the pharmacophore contribute equally to binding. Thus, for example, epimerization of the C-26 hydroxyl of bryostatin 4 (which is homologous to the C-20 hydroxyl of phorbol) caused complete loss of binding affinity under comparable binding conditions.32 Another important aspect of the biological evaluations of bryostatins 16-18 concerned effects against the P388 lymphocytic leukemia and a minipanel of the U. S. National Cancer Institute's human cancer cell lines.33 The GI50 (µg/mL) for bryostatin 16 ranged from 0.019 to 2.2, for bryostatin 17, 0.16 to 3.2, and for bryostatin 18, 0.42 to 3.7. In each case the greatest activity was displayed against the lung (NCI-H460) cell line. Specifically, bryostatin 16 resulted in inhibitory activity against ovarian (OVCAR-3: GI50 1.9 µg/mL), CNS (SF-295: GI50 1.4 µg/mL), renal (A498: GI50 1.9 µg/mL), lung (NCI-H460: GI50 0.019 µg/mL), colon (KM20L2: GI50 2.2 µg/mL), and melanoma (SK-MEL-5: GI50 1.2 µg/mL). Bryostatins 17 and 18 were evaluated against the same minipanel with very similar results. Experimental Section General Experimental Procedures. Solvents used for column chromatography were redistilled. Sephadex

bryostatin 10 bryostatin 16 bryostatin 17 bryostatin 18 C-1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 OR 1′ 2′ 3′-5′

172.34 42.72 69.82 40.93 66.70 34.02 73.15 42.24 102.05 42.72 73.42 44.70 158.76 37.32 80.53 130.91 139.77 45.56 102.73 40.39 158.53 36.63 65.76 36.11 73.42 69.07 19.08 20.46 17.34 114.53 167.70 21.32 24.71 116.23 167.47 51.60 51.40

172.80 42.92 67.41 42.54 65.97 34.31 73.48 41.86 102.50 43.04 74.42 45.29 158.95 36.48 79.94 128.20 139.49 42.07 151.91 101.55 168.22 32.82 73.48 36.48 73.48 69.02 19.14 21.29 17.23 114.82 167.78 27.72 23.45 108.90 170.02 51.59 51.21

174.85 42.73 65.77 42.64 69.01 34.00 75.09 42.11 101.71 37.08 75.97 43.75 157.68 37.10 81.27 127.45 140.70 43.75 149.80 97.14 167.97 37.10 74.37 36.26 73.35 69.02 19.36 21.37 15.28 115.50 167.61 27.93 22.64 108.89 170.23 51.58 51.29

172.37 42.73 69.81 40.95 66.70 34.02 73.20 42.26 102.75 42.73 73.44 44.71 158.89 37.36 80.51 130.94 139.85 45.68 101.98 33.70 158.81 43.13 66.17 36.03 73.25 69.08 19.18 20.55 17.35 114.52 167.73 21.33 24.75 116.26 167.67 51.61 51.40

178.49 39.64 27.45

178.51 39.65 27.47

178.42 39.67 27.46

178.54 39.67 27.46

LH-20 was obtained from Pharmacia Fine Chemicals AB. All 1H- and 13C-NMR data were obtained using a Bruker AM 400 spectrometer. Optical rotations were measured using a Perkin-Elmer Model 241 polarimeter. The IR spectra were recorded using a Nicolet MX-1 FTIR spectrometer. UV detection at 254 nm was used for HPLC. For other general experimental procedures refer to ref 10. Animal Material. An approximate 1000 kg (wet wt) amount of B. neritina L. was collected in May 1986 in the Northern Gulf of Mexico (Florida) and preserved in 2-propanol. A voucher specimen of B. neritina is maintained at the Arizona State University Cancer Research Institute. For animal taxonomy and earlier recollection data, see ref 4. Isolation. The biomass was reduced to a murine P388 lymphocytic leukemia (PS) active fraction weighing 906 g as previously described.10 This fraction was further separated as summarized for isolation of bryostatin 14.10 The two PS active and complex fractions from the high speed countercurrent distribution step were separated as follows. The fraction containing bryostatins 16 and 17 was separated by reversed-phase semipreparative HPLC using an RP C-8 column (10 mm × 25 cm) with 4:1 CH3OH-H2O as eluting solvent (1.0 mL/min). Bryostatin 16 (4.3 mg) was eluted at 25.7 min and bryostatin 17

Notes

(3.4 mg) at 18.0 min. Separation of the fraction containing bryostatin 18 was accomplished by normalphase silica gel semipreparative HPLC (10.0 mm × 25 cm column) using hexane-2-propanol (9:1) as eluant. Bryostatin 18 (6.7 mg) eluted at 26 min. The known bryostatins 4-8 and bryostatin 10 were also isolated from this fraction. Bryostatin 16: [R]D ) +84° (c ) 0.43, CH3OH); LRFABMS (3-NBA/glycerol matrix) m/z (rel int), 813 [M + Na]+ (10), 790 [M]+ (30), 773 [M - OH]+ (80), 741 [773 - CH3OH]+ (100); HRFABMS (C42H62O14Na, calcd 813.4037) found 813.4038; NMR data, see Tables 1 and 2. Bryostatin 17: [R]D ) +231° (c ) 0.34, CH3OH); LRFABMS (3-NBA/glycerol matrix) m/z (rel int), 813 [M + Na]+ (4), 790 [M]+ (29), 773 [M - OH]+ (100), 741 [773 - CH3OH]+ (78); LRFABMS (3-NBA/LiCl matrix) m/z (rel int) 797 [M + Li]+ (100); HRFABMS (C42H62O14Li, calcd 797.4277) found 797.4301; NMR data, see Tables 1 and 2. Bryostatin 18: [R]D ) +136° (c ) 0.67, CH3OH); IR (film) νmax 3465, 3370 (OH), 1740 (COOR), 1650 (CdC), 1610 (conjugated CdC), 1380 (CH3), 1155 (COOR), 860 (CH3); LRFABMS (3-NBA/LiCl matrix) m/z (rel int), 815 [M + Li]+ (100), 797 [M + Li - H2O]+ (42); HRFABMS (C42H64O15Li calcd 815.4405), found 815.4432; NMR data, see Tables 1 and 2. Acknowledgments. With appreciation we acknowledge Outstanding Investigator Grant CA 44344-01A106 and Grant CA-16049-05-12 with the Division of Cancer Treatment, NCI, DHHS, the Arizona Disease Control Research Commission, the Fannie E. Rippel Foundation, the Robert B. Dalton Endowment, Virginia Piper, Diane Cummings Halle, Polly J. Trautman, and Lotte Flugel for the very necessary financial support. For other assistance we are also pleased to thank Drs. Dennis L. Doubek, John R. Van Camp, and Fiona Hogan and Mr. Jack J. Rudloe and the NSF Regional Instrumentation Facility in Nebraska (Grant CHE-8620177). References and Notes (1) Part 339: Pettit, G. R. The Dolastatins. In Progress in the Chemistry of Organic Natural Products, submitted. (2) Pettit, G. R. The Bryostatins. In Progress in the Chemistry of Organic Natural Products; Herz, W., Kirby, G. W., Steglich, W., Tamm, C., Eds.: Springer-Verlag: New York, 1991; No. 57, pp 153-195. (3) Pettit, G. R.; Day, J. F.; Hartwell, J. L.; Wood, H. G. Nature 1970, 227, 962-963. (4) Pettit, G. R.; Kamano, Y.; Herald, C. L.; Tozawa, M. J. Am. Chem. Soc. 1984, 106, 6768-6771. (5) Pettit, G. R.; Kamano, Y.; Herald, C. L.; Tozawa, M. Can. J. Chem. 1985, 63, 1204-1208.

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(6) Pettit, G. R.; Kamano, Y.; Aoyagi, R.; Herald, C. L.; Doubek, D. L.; Schmidt, J. M.; Rudloe, J. J. Tetrahedron 1985, 41, 985994. (7) Pettit, G. R.; Kamano, Y.; Herald, C. L. J. Nat. Prod. 1986, 49, 661-664. (8) Pettit, G. R.; Kamano, Y.; Herald, C. L. J. Org. Chem. 1987, 52, 2848-2854. (9) Pettit, G. R.; Leet, J. E.; Herald, C. L.; Kamano, Y.; Boettner, F.; Baczynskyj, L.; Nieman, R. A. J. Org. Chem. 1987, 52, 28542860. (10) Pettit, G. R.; Gao, F.; Sengupta, D.; Coll, J. C.; Herald, C. L.; Doubek, D. L.; Schmidt, J. M.; Van Camp, J. R.; Rudloe, J. J.; Nieman, R. A. Tetrahedron 1991, 47, 3601-3610. (11) Jayson, G. C.; Crowther, D.; Prendiville, J.; McGown, A. T.; Scheid, C.; Stern, P.; Young, R.; Brenchley, P.; Chang, J.; Owens, S.; Pettit, G. R. Br. J. Cancer 1995, 72, 461-468. (12) Scheid, J.; Prendiville, J.; Jayson, G.; Crowther, D.; Fox, B.; Pettit, G. R.; Stern, P. L. Cancer Immunology and Immunotherapy 1994, 39, 223-230. (13) Philip, P. A.; Rea, D.; Thavasu, P.; Carmichael, J.; Stuart, N. S. A.; Rockett, H.; Talbot, D.; Ganesan, T.; Pettit, G. R.; Balkwill, F.; Harris, A. L. J. Natl. Cancer Inst. 1993, 85, 1812-1818. (14) Prendiville, J.; Crowther, D.; Thatcher, N.; Woll, P. J.; Fox, B. W.; McGown, A.; Testa, N.; Stern, P.; McDermott, R.; Potter, M.; Pettit, G. R. Br. J. Cancer 1993, 68, 418-424. (15) Szallasi, Z.; Denning, M. F.; Smith, C. B.; Dlugosz, A. A.; Yuspa, S. H.; Pettit, G. R.; Blumberg, P. M. Molecular Pharm. 1994, 46, 840-850. (16) Stanwell, C.; Gescher, A.; Bradshaw, T.; Pettit, G. R. Int. J. Cancer 1994, 56, 585-592. (17) Grant, S.; Traylor, R.; Pettit, G. R.; Lin, P.-S. Blood 1994, 83, 663-667. (18) Grant, S.; Traylor, R.; Pettit, G. R. Lin, P.-S. Cytokine 1993, 5, 490-497. (19) Berkow, R. L.; Schlabach, L.; Dodson, R.; Benjamin, W. H.; Pettit, G. R.; Rustagi, P.; Kraft, A. S. Cancer Res. 1993, 53, 2810-2815. (20) Al-Katib, A.; Mohammad, R. M.; Khan, K.; Dan, M. E.; Pettit, G. R.; Sensenbrenner, L. L. J. Immunother. 1993, 14, 33-42. (21) McCrady, C. W.; Li, F.; Pettit, G. R.; Grant, S. Exp. Hematol. 1993, 21, 893-900. (22) Isakov, N.; Galron, D.; Mustelin, T.; Pettit, G. R.; Altman, A. J. Immunol. 1993, 150, 1195-1204. (23) Pettit, G. R.; Sengupta, D.; Blumberg, P. M.; Lewin, N. E.; Schmidt, J. M.; Kraft, A. S. Anti-Cancer Drug Des. 1992, 7, 101113. (24) Pettit, G. R.; Sengupta, D.; Herald, C. L.; Sharkey, N. A.; Blumberg, P. M. Can. J. Chem. 1991, 69, 856-860. (25) For other recent advances in isolation of bryozoan constituents see: Kamano, Y.; Zhang, H-P.; Hino, A.; Yoshida, M.; Pettit, G. R.; Herald, C. L.; Itokawa, H. J. Nat. Prod., submitted. (26) Kantoci, D.; Pettit, G. R.; Cichacz, Z. A. J. Liquid Chromatogr. 1991, 14, 1149-1160. (27) Wender, P. A.; Cribbs, C. M.; Koehler, K. F.; Sharkey, N. A.; Herald, C. L.; Kamano, Y.; Pettit, G. R.; Blumberg, P. M. Proc. Natl. Acad. Sci. U.S.A. 1988, 85, 7197-7201. (28) Wender, P. A.; Koehler, K. F.; Sharkey, N. A.; Dell’Aquila, M. L.; Blumberg, P. M. Proc. Natl. Acad. Sci. U.S.A. 1986, 83, 42144218. (29) Rando, R.; Kishi, Y. Biochemistry 1992, 31, 2211-2218. (30) Lewin, N. E.; Dell’Aquila, M. L.; Pettit, G. R.; Blumberg, P. M.; Warren, B. S. Biochem. Pharmacol. 1992, 43, 2007-2014. (31) Kazanietz, M. G.; Lewin, N. E.; Gao, F.; Pettit, G. R.; Blumberg, P. M. Mol. Pharmacol. 1994, 46, 374-379. (32) Lewin, N. E.; Pettit, G. R.; Kamano, Y.; Blumberg, P. M. Cancer Commun. 1991, 3, 67-70. (33) Boyd, M. R. In Current Therapy in Oncology; Neiderhuber, J. E., Ed.; B. C. Decker: Philadelphia, 1992, pp 11-22.

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